Lipase inhibiting polymers

ABSTRACT

The invention features a method for treating obesity in a patient by administering to the patient a polymer that has been substituted with one or more groups that inhibit lipases, which are enzymes responsible for the hydrolysis of fat. The invention further relates to the polymers employed in the methods described herein as well as novel intermediates and methods for preparing the polymers.

RELATED APPLICATION

This application is a Divisional of U.S. application Ser. No.09/714,541, filed Nov. 16, 2000, which is a Divisional of U.S.application Ser. No. 09/226,585, filed Jan. 6, 1999, now U.S. Pat. No.6,352,692, which is a Continuation-in-Part of U.S. application Ser. No.09/166,510 filed Oct. 5, 1998, now U.S. Pat. No. 6,267,952, which is aContinuation-in-Part of U.S. application Ser. No. 09/005,379 filed onJan. 9, 1998, now abandoned, the entire teachings of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Human obesity is a recognized health problem with approximatelyninety-seven million people considered clinically overweight in theUnited States. The accumulation or maintenance of body fat bears adirect relationship to caloric intake. Therefore, one of the most commonmethods for weight control to combat obesity is the use of relativelylow-fat diets, that is, diets containing less fat than a “normal diet”or that amount usually consumed by the patient.

The presence of fats in a great many food sources greatly limits thefood sources which can be used in a low fat diet. Additionally, fatscontribute to the flavor, appearance and physical characteristics ofmany foodstuffs. As such, the acceptability of low-fat diets and themaintenance of such diets are difficult.

Various chemical approaches have been proposed for controlling obesity.Anorectic agents such as dextroamphetamine, the combination of thenon-amphetamine drugs phentermine and fenfluramine (Phen-Fen), anddexfenfluramine (Redux) alone, are associated with serious side effects.Indigestible materials such as olestra (OLEAN®), mineral oil orneopentyl esters (see U.S. Pat. No. 2,962,419) have been proposed assubstitutes for dietary fat. Garcinia acid and derivatives thereof havebeen described as treating obesity by interfering with fatty acidsynthesis. Swellable crosslinked vinyl pyridine resins have beendescribed as appetite suppressants via the mechanism of providingnon-nutritive bulk, as in U.S. Pat. No. 2,923,662. Surgical techniquessuch as temporary ileal bypass surgery, are employed in extreme cases.

However, methods for treating obesity, such as those described abovehave serious shortcomings with controlled diet remaining the mostprevalent technique for controlling obesity. As such, new methods fortreating obesity are needed.

SUMMARY OF THE INVENTION

The invention features a method for treating obesity in a patient byadministering to the patient a polymer that has been substituted with orcomprises one or more groups which can inhibit a lipase. Lipases are keyenzymes in the digestive system which break down tri- and diglycerides,which are too large to be absorbed by the small intestine into fattyacids which can be absorbed. Therefore, inhibition of lipases results ina reduction in the absorption of fat. In one embodiment, the lipaseinhibiting group can be a “suicide substrate” which inhibits theactivity of the lipase by forming a covalent bond with the enzyme eitherat the active site or elsewhere. In another embodiment, the lipaseinhibiting group is an isosteric inhibitor of the enzyme. The inventionfurther relates to the polymers employed in the methods described hereinas well as novel intermediates and methods for preparing the polymers.

DETAILED DESCRIPTION OF THE INVENTION

The invention features a method for treating obesity in a patient byadministering to the patient a polymer comprising one or more groupswhich can inhibit a lipase. Since lipases are responsible for thehydrolysis of fat, a consequence of their inhibition is a reduction infat hydrolysis and absorption. The invention further relates to thepolymers employed in the methods described herein as well as novelintermediates and methods for preparing the polymers.

In one aspect of the invention, the lipase inhibiting group inactivatesa lipase such as gastric, pancreatic and lingual lipases. Inactivationcan result by forming a covalent bond such that the enzyme is inactive.The covalent bond can be formed with an amino acid residue at or nearthe active site of the enzyme, or at a residue which is distant from theactive site provided that the formation of the covalent bond results ininhibition of the enzyme activity. Lipases contain a catalytic triadwhich is responsible for the hydrolysis of lipids into fatty acids. Thecatalytic triad consists of a serine, aspartate and histidine amino acidresidues. This triad is also responsible for the hydrolysis of amidebonds in serine proteases, and it is expected that compounds that areserine protease inhibitors will also inhibit lipases. Therefore, serineprotease inhibitors that can be covalently linked to a polymer arepreferred lipase inhibiting groups. For example, a covalent bond can beformed between the lipase inhibiting group and a hydroxyl at or thecatalytic site of the enzyme. For instance, a covalent bond can beformed with serine. Inactivation can also result from a lipaseinhibiting group forming a covalent bond with an amino acid, for examplecysteine, which is at some distance from the active site. In addition,non-covalent interaction between the lipase inhibiting group and theenzyme can also result in inactivation of the enzyme. For example, thelipase inhibiting group can be an isostere of a fatty acid, which caninteract non-covalently with the catalytic site of the lipase. Inaddition, the lipase inhibiting group can compete for lipase hydrolysiswith natural triglycerides.

In one aspect of the invention, a lipase inhibiting group can berepresented by formula I:

wherein,

-   -   R is a hydrogen, hydrophobic moiety, —NR²R³, —CO₂H, —OCOR²,        —NHCOR², a substituted or unsubstituted aliphatic group or a        substituted or unsubstituted aromatic group;    -   R¹ is an activating group;    -   Y is oxygen, sulfur, —NR²— or is absent;    -   Z and Z¹ are, independently, an oxygen, alkylene, sulfur, —SO₃—,        —CO₂—, —NR₂—, —CONR²—, —PO₄H— or a spacer group;    -   R² and R³ are, independently, a hydrogen, a substituted or        unsubstituted aliphatic group, or a substituted or unsubstituted        aromatic group;    -   m is 0 or 1; and    -   n is 0 or 1.

In one embodiment, the lipase inhibiting group of formula I can berepresented by the following structures:

wherein R, R¹ and Y are defined as above.

In another embodiment, the lipase inhibiting group of structural formulaI can be represented by the following structures:

wherein R, R¹, R², R³ and Y are defined as above, and p is an integer(e.g. an integer between zero and about 30, preferably between about 2and about 10).

In another embodiment, the lipase inhibitor of formula I is a mixedanhydride. Mixed anhydrides include, but are not limited to,phosphoric-carboxylic, phosphoric-sulfonic and pyrophosphate mixedanhydride lipase inhibiting groups which can be represented by thefollowing structures, respectively:

wherein R, R¹, Y and Z¹ are defined as above.

In another aspect, a lipase inhibiting group of the invention can be ananhydride. In one embodiment, the anhydride is a cyclic anhydriderepresented by formula II:

wherein R, Z and p are defined as above, X is —PO₂—, —SO₂— or —CO—, andk is an integer from 1 to about 10, preferably from 1-4.

In another embodiment, the anhydride lipase inhibiting groups can be acyclic anhydride which is part of a fused ring system. Anhydrides ofthis type can be represented by formula III:

wherein X and Z are defined as above, and ring A is an optionallysubstituted cyclic aliphatic group or aromatic group, or combinationsthereof, which can include one or more heteroatoms in the ring. In aparticular embodiment, the cyclic anhydride is a benzenesulfonicanhydride represented by the following structure:

wherein Z is defined as above and the benzene ring can be furthersubstituted.

In another aspect, the lipase inhibiting group is an α-halogenatedcarbonyl which can be represented by formula IV:

wherein R and Y are defined as above, and W¹ and W² are eachindependently hydrogen or halogen, for example, —F, —Cl, —Br, and —I,wherein at least one of W¹ and W² is a halogen.

In yet another aspect, a cyclic compound having an endocyclic group thatis susceptible to nucleophilic attack can be a lipase inhibiting group.Lactones and epoxides are examples of this type of lipase inhibitinggroup and can be represented by formulas V and VI, respectively:

wherein R, Z, m and p are defined as above.

In a further aspect, the lipase inhibiting group can be a sulfonate ordisulfide group represented by formulas VII and VIII, respectively:

wherein R, Z and p are defined as above, and R⁵ is absent or ahydrophobic moiety, a substituted or unsubstituted aliphatic group or asubstituted or unsubstituted aromatic group.

In a particular embodiment, the disulfide lipase inhibiting group can berepresent by the following formula:

wherein R, Z and p are defined as above.

In a further aspect of the invention, a lipase inhibiting group can be aboronic acid which can be linked to a polymer by a hydrophobic group orto the polymer directly when the polymer is hydrophobic. Boronic acidlipase inhibiting groups can be represented by the following structure:

wherein R⁵, Z, n and m are defined as above.

In an additional aspect, an isosteric lipase inhibiting group can be aphenolic acid linked to the polymer. Phenolic acid lipase inhibitinggroups can be represented by the following structure:

wherein Z, R⁵, n and m are defined as above and —CO₂H and —OH are orthoor para with respect to each other.

A variety of polymers can be employed in the invention described herein.The polymers can be aliphatic, alicyclic or aromatic or synthetic ornaturally occurring. However, aliphatic and alicyclic synthetic polymersare preferred. Furthermore, the polymer can be hydrophobic, hydrophilicor copolymers of hydrophobic and/or hydrophilic monomers. The polymercan be non-ionic (e.g., neutral), anionic or cationic, in whole or inpart. Furthermore, the polymers can be manufactured from olefinic orethylenic monomers (such as vinylalcohol) or condensation polymers.

For example, the polymers can be a polyvinylalcohol, polyvinylamine,poly-N-alkylvinylamine, polyallylamine, poly-N-alkylallylamine,polyalkylenimine, polyethylene, polypropylene, polyether, polyethyleneoxide, polyamide, polyacrylic acid, polyalkylacrylate, polyacrylamide,polymethacrylic acid, polyalkylmethacrylate, polymethacrylamide,poly-N-alkylacrylamide, poly-N-alkylmethacrylamide, polystyrene,vinylnaphthalene, ethylvinylbenzene, aminostyrene, vinylbiphenyl,vinylanisole, vinylimidazolyl, vinylpyridinyl,dimethylaminomethylstyrene, trimethylammoniumethylmethacrylate,trimethylammoniumethylacrylate, carbohydrate, protein and substitutedderivatives of the above (e.g., fluorinated monomers thereof) andcopolymers thereof.

Preferred polymers include polyethers, such as polyalkylene glycols.Polyethers can be represented by the formula IX:

wherein R is defined as above and q is an integer.

For example, the polymer can be polypropylene glycol or polyethyleneglycol or copolymers thereof. The polymers can be random or blockcopolymers. Also, the polymers can be hydrophobic, hydrophilic, or acombination thereof (as in random or block polymers).

A particularly preferred polymer is a block copolymer characterized byhydrophobic and hydrophilic polymeric regions. In such an embodiment,the “core polymer can be hydrophobic with one or both ends capped with ahydrophilic polymer or vice versa. An example of such a polymer is apolyethyleneglycol-polypropyleneglycol-polethyleneglycol copolymer, asis sold under the tradename PLURONIC® (BASF Wyandotte Corp.). BRIJ® andIGEPAL® (Aldrich, Milwaukee, Wis.) are examples of polymers having apolyethylene glycol core capped withe a hydrophobic end group. BRIJ®polymers are polyethylene glycols having one end capped with alkoxygroup, while the hydroxy group at the other end of the polymer chain isfree. IGEPAL® polymers are polyethylene glycols having one end cappedwith 4-nonylphenoxy group, while the hydroxy group at the other end ofthe polymer chain is free.

Another class of polymers includes aliphatic polymers such as,polyvinylalcohol, polyallylamine, polyvinylamine and polyethylenimine.These polymers can be further characterized by one or more substituents,such as substituted or unsubstituted, saturated or unsaturated alkyl andsubstituted or unsubstituted aryl. Suitable substituents includeanionic, cationic or neutral groups, such as alkoxy, aryl, aryloxy,aralkyl, halogen, amine, and ammonium groups, for example. The polymercan desirably possess one or more reactive functional groups which can,directly or indirectly, react with an intermediate possessing the lipaseinhibiting groups.

In one embodiment, the polymers have the following repeat unit:

wherein,

-   -   q is an integer; and    -   R⁴ is —OH, —NH₂, —CH₂NH₂, —SH, or a group represented by the        following formula:        wherein R, R¹, Y, Z, Z¹, m and n are defined as above.

Additionally, the polymer can be a carbohydrate, such as chitosan,cellulose, hemicellulose or starch or derivatives thereof.

The polymer can be linear or crosslinked. Crosslinking can be performedby reacting the copolymer with one or more crosslinking agents havingtwo or more functional groups, such as electrophilic groups, which reactwith an alcohol of the polymer to form a covalent bond. Crosslinking inthis case can occur, for example, via nucleophilic attack of the polymerhydroxy groups on the electrophilic groups. This results in theformation of a bridging unit which links two or more alcoholic oxygensfrom different polymer strands. Suitable crosslinking agents of thistype include compounds having two or more groups selected from amongacyl chloride, epoxide, and alkyl-X, wherein X is a suitable leavinggroup, such as a halo, tosyl or mesyl group. Examples of such compoundsinclude, but are not limited to, epichlorohydrin, succinyl dichloride,acryloyl chloride, butanedioldiglycidyl ether, ethanedioldiglycidylether, pyromellitic dianhydride, and dihaloalkanes.

The polymer composition can also be crosslinked by including amultifunctional co-monomer as the crosslinking agent in the reactionmixture. A multifunctional co-monomer can be incorporated into two ormore growing polymer chains, thereby crosslinking the chains. Suitablemultifunctional co-monomers include, but are not limited to,diacrylates, triacrylates, and tetraacrylates, dimethacrylates,diacrylamides, diallylacrylamides, and dimethacrylamides. Specificexamples include ethylene glycol diacrylate, propylene glycoldiacrylate, butylene glycol diacrylate, ethylene glycol dimethacrylate,butylene glycol dimethacrylate, methylene bis(methacrylamide), ethylenebis(acrylamide), ethylene bis(methacrylamide), ethylidenebis(acrylamide), ethylidene bis(methacrylamide), pentaerythritoltetraacrylate, trimethylolpropane triacrylate, bisphenol Adimethacrylate, and bisphenol A diacrylate. Other suitablemultifunctional monomers include polyvinylarenes, such asdivinylbenzene.

The molecular weight of the polymer is not critical. It is desirablethat the polymer be large enough to be substantially or completelynon-absorbed in the GI tract. For example, the molecular weight can bemore than 900 Daltons.

The digestion and absorption of lipids is a complex process in whichwater insoluble lipids are emulsified to form an oil in water emulsionwith an oil droplet diameter of approximately 0.5 mm. This emulsifiedoil phase has a net negative charge due to the presence of fatty acidsand bile salts, which are the major emulsifying agents. Lipases that arepresent in the aqueous phase hydrolyze the emulsified lipids at theemulsion surface. Most lipases contain an active site that is buried bya surface loop of amino acids that sit directly on top of the activesite when the lipase is in an aqueous solution. However, when the lipasecomes in contact with bile salts at the lipid/water interface of a lipidemulsion, the lipase undergoes a conformational change that shifts thesurface loop to one side and exposes the active site. Thisconformational change allows the lipase to catalyze hydrolysis of lipidsat the lipid/water interface of the emulsion. Polymers that disrupt thesurface of the emulsion or alter its chemical nature are expected toinhibit lipase activity. Therefore, it may increase the effectiveness ofpolymers that have lipase inhibiting groups to administer them with oneor more polymers that alter the emulsion surface. Alternatively, lipaseinhibiting groups can be attached directly to such a polymer.

Several types of fat-binding polymers have been effective in disruptingthe surface of the lipid emulsion or altering its chemical nature. Forexample, polymers that have positively charged emulsifiers are able toform stable polycation lipid emulsions. The lipids in such an emulsionare not substrates for gastrointestinal lipases because the surface ofthe emulsion has a net positive charge instead of the usual net negativecharge. Another type of fat-binding polymer destabilizes the emulsioncausing the oil droplets of the emulsion to coalesce. This decreases theemulsion surface area where lipases are active, and therefore, reduceslipid hydrolysis. Fat-binding polymer are further defined in copendingapplication Ser. No. 09/004,963, filed on Jan. 9, 1998, and applicationSer. No.09/166,453, filed on Oct. 5, 1998, the contents of which areincorporated herein by reference.

The substituted polymers described herein can be manufactured accordingto methods generally known in the art. For example, a lipase inhibitingintermediate characterized by a reactive moiety can be contacted with apolymer characterized by a functional group which reacts with saidreactive moiety. See March, J., Advanced Organic Chemistry, 3^(rd)edition, John Wiley and Sons, Inc.; New York, (1985).

A “hydrophobic moiety,” as the term is used herein, is a moiety which,as a separate entity, is more soluble in octanol than water. Forexample, the octyl group (C₈H₁₇) is hydrophobic because its “parent”alkane, octane, has greater solubility in octanol than in water. Thehydrophobic moieties can be a saturated or unsaturated, substituted orunsubstituted hydrocarbon group. Such groups include substituted andunsubstituted, normal, branched or cyclic aliphatic groups having atleast four carbon atoms, substituted or unsubstituted arylalkyl orheteroarylalkyl groups and substituted or unsubstituted aryl orheteroaryl groups. Preferably, the hydrophobic moiety includes analiphatic group of between about six and thirty carbons. Specificexamples of suitable hydrophobic moieties include the following alkylgroups: butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl,tetradecyl, hexadecyl, octadecyl, docosanyl, cholesteryl, famesyl,aralkyl, phenyl, and naphthyl, and combinations thereof. Other examplesof suitable hydrophobic moieties include haloalkyl groups of at leastfourcarbons (e.g., 10-halodecyl), hydroxyalkyl groups of at least sixcarbons (e.g., 11-hydroxyundecyl), and aralkyl groups (e.g., benzyl). Asused herein aliphatic groups include straight, chained, branched orcyclic C₄-C₃₀ hydrocarbons which are completely saturated or contain oneor more units of unsaturation.

Aromatic groups suitable for use in the invention include, but are notlimited to, aromatic rings, for example, phenyl and substituted phenyl,heteroaromatic rings, for example, pyridinyl, furanyl and thiophenyl,and fused polycyclic aromatic ring systems in which a carbocyclicaromatic ring or heteroaryl ring is fused to one or more othercarbocyclic or heteroaryl rings. Examples of fused polycyclic aromaticring systems include substituted or unsubstituted phenanthryl,anthracyl, naphthyl, 2-benzothienyl, 3-benzothienyl, 2-benzofuranyl,3-benzofuranyl, 2-indolyl, 3-indolyl, 2-quinolinyl, 3-quinolinyl,2-benzothiazole, 2-benzooxazole, 2-benzimidazole, 2-quinolinyl,3-quinolinyl, 1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl, 3-isoindolyl,and acridintyl.

A “substituted aliphatic or aromatic group” can have one or moresubstituents, e.g., an aryl group (including a carbocyclic aryl group ora heteroaryl group), a substituted aryl group, —O-(aliphatic group oraryl group), —O-(substituted aliphatic group or substituted aryl group),acyl, —CHO, —CO-(aliphatic or substituted aliphatic group), —CO-(aryl orsubstituted aryl), —COO-(aliphatic or substituted aliphatic group),—COO-(aryl or substituted aryl group), —NH-(acyl), —O-(acyl), benzyl,substituted benzyl, halogenated lower alkyl (e.g. trifluoromethyl andtrichloromethyl), fluoro, chloro, bromo, iodo, cyano, nitro, —SH,—S-(aliphatic or substituted aliphatic group), —S-(aryl or substitutedaryl), —S-(acyl) and the like.

An “activating group” is a group that renders a functional group ormoiety reactive. Generally, electron withdrawing groups are “activatinggroups.” R¹ or Y—R¹, of the above formulae, is preferably a good leavinggroup or an electron withdrawing group. Examples of good leaving groupsare phosphate, p-nitrophenol, o,p-dinitrophenol, N-hydroxysuccinimide,imidazole, ascorbic acid, pyridoxine, trimethylacetate,adamantanecarbonylate, p-chlorophenol, o,p-dichlorophenol,methanesulfonylate, mesitylsulfonylate andtriisopropylbenzenesulfonylate. A preferred leaving group isN-hydroxysuccinimide.

A spacer group can be a group that has one to about thirty atoms and iscovalently bonded to the lipase inhibitor, to the polymer, or to thehydrophobic moiety. Generally, the spacer group can be covalently bondedto the lipase inhibitor, polymer or hydrophobic moiety through afunctional group. Examples of functional groups are oxygen, alkylene,sulfur, —SO₂—, —CO₂—, —NR₂—, or —CONR²—. A spacer group can behydrophilic or hydrophobic. Examples of spacer groups include aminoacids, polypeptides, carbohydrates, and optionally substituted alkyleneor aromatic groups. Spacer groups can be manufactured from, for example,epichlorohydrin, dihaloalkane, haloalkyl esters, polyethylene glycol,polypropylene glycol and other cross-linking or difunctional compounds.Bromoalkylacetate is a preferred spacer group.

The amount of a polymer administered to a subject will depend on thetype and severity of the disease and on the characteristics of thesubject, such as general health, age, body weight and tolerance todrugs. It will also depend on the degree of obesity and obesity relatedcomplications. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. Typically, in humansubjects, an effective amount of the polymer can range from about 10 mgper day to about 50 mg per day for an adult. Preferably, the dosageranges from about 10 mg per day to about 20 mg per day.

The polymer can be administered by any suitable route, including, forexample, orally in capsules, suspensions or tablets. Oral administrationby mixing with food is a preferred mode of administration.

The polymer can be administered to the individual in conjunction with anacceptable pharmaceutical carrier as part of a pharmaceuticalcomposition. Formulation of a polymer to be administered will varyaccording to the route of administration selected (e.g., solution,emulsion, capsule). Suitable pharmaceutical carriers may contain inertingredients which do not interact with the lipase inhibiting groups ofthe polymer. Standard pharmaceutical formulation techniques can beemployed, such as those described in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. Methods for encapsulatingcompositions (such as in a coating of hard gelatin or cyclodextran) areknown in the art (Baker, et al., “Controlled Release of BiologicalActive Agents”, John Wiley and Sons, 1986).

Experimental

Synthesis of Polymers

EXAMPLE 1 Preparation of Polyethylene Glycol having an n-pentylHydroprobic Moiety and p-nitrophenyl Phosphate Lipase Inhibiting Groups

A mixture of n-pentanol (19.5 mmol. 1.72 g) and N-methyl imidazole (19.5mmol, 1.6 g) in anhydrous methylene chloride (40 mL) was added slowlyover 20 minutes under anhydrous conditions to a solution ofp-nitrophenyl phosphorodichloridate (5.0 g, 19.5 mmol) in anhydrousmethylene chloride (100 mL). The reaction flask was cooled in a waterbath during the addition. After the completion of the addition, thewater bath was removed, and the reaction mixture was stirred for 2 hoursat room temperature. A mixture of polyethyleneglycol (MW=8,000; 10 mmol,80 g), and N-methyl imidazole (19.5 mmol, 1.6 g) in anhydrous methylenechloride (150 mL) was added to the reaction flask under anhydrousconditions. The mixture was stirred for 25 hours at room temperature.The solvent was removed under vacuum, the residue was purified accordingto method A, and the polymer was obtained as white powder (70 g).

Purification Procedures

Method A:

The residue was dissolved in de-ionized water (100 mL). The solution wasdialyzed for 24 hours using Spectra/Por Membrane MWCO: 3,500. Thedialyzed solution was lyophilized, and the polymer was obtained as whitepowder.

Method B:

The residue was poured into 0.5 L of diethyl ether and stirred at roomtemperature for 1 hour. The solvent was decanted and replaced with freshdiethyl ether (0.25 L). The mixture was stirred for 1 hour. The solventwas removed, and the polymer was dried at room temperature under vacuum.

Method C:

The reaction mixture was washed with 10% aqueous sodium sulfate solution(3×100 mL). The organic phase was dried over magnesium sulfate. Thesolvent was removed, and the polymer was dried at room temperature.

Using the above procedures, the following compounds were synthesized andare tabulated in the following table. TABLE 1 Polyethylene glycols (PEG)having p-nitrophenyl phosphate lipase inhibiting groups with a varietyof hydroprobic moieties. PEG HYDROPHOBIC METHOD OF PHYSICAL EXAMPLE MWMOIETY (R) PURIFICATION STATE 1 8,400 n-pentyl Method A powder 2 3,400n-decyl Method B powder 3 3,400 n-dodecyl Method B powder 4 3,400n-octadecyl Method B powder 5 1,000 n-decyl Method B semi solid 6 1,000n-dodecyl Method B semi solid 7 1,000 n-tetradecyl Method B semi solid 81,000 n-hexadecyl Method B semi solid 9 1,000 n-octadecyl Method B semisolid 10 1,000 n-pentyl Method C semi solid 11 1,000 n-hexyl Method Csemi solid 12 1,000 n-octyl Method C semi solid 13 1,000 n-docosylMethod C powder 14 1,000 cholesteryl Method C powder 15 3,400 n-pentylMethod B solid 16 1,500 n-pentyl Method B solid 17 1,500 n-decyl MethodB solid 18 1,500 n-dodecyl Method B solid 19 1,500 n-hexadecyl Method Csolid 20 1,500 n-octadecyl Method C solid 21 1,500 n-docosyl Method Bsolid 22 1,500 rac-farnesyl Method B brown, solid 23 1,500 n-cholesterylMethod C solid 24 1,500 5-phenyl-1-pentyl Method C solid 25 1,500n-octyl Method C solid 26 1,500 n-hexyl Method C solid 27 3,400 n-octylMethod C solid 28 8,400 n-octyl Method C solid

EXAMPLE 29 Preparation of a PLURONIC® Polymer having a n-tetradecylHydrophobic Moiety and p-nitrophenyl Phosphate Lipase Inhibiting Groups

A mixture of n-tetradecanol (15 g, 70 mmol) and N-methyl imidazole (5.6mL, 70 mmol) in anhydrous methylene chloride (75 mL) was added slowlyover 20 minutes under anhydrous condition to a solution of p-nitrophenylphosphorodichloridate (17.92 g, 70 mmol) in anhydrous methylene chloride(50 mL). The reaction flask was cooled in a water bath during theaddition. After the completion of the addition, the water bath wasremoved, and the reaction mixture was stirred for 2 hours at roomtemperature. A mixture of PLURONIC® (MW=1,100; 39 g, 35 mmol) andN-methyl imidazole (5.6 mL. 70 mmol) in anhydrous methylene chloride(150 mL) was added to the reaction flask under anhydrous conditions. Themixture was stirred for 24 hours at room temperature. The reactionmixture was extracted with cold saturated NaCl solution (3×150 mL), theorganic layer was dried over anhydrous sodium sulfate. The sodiumsulfate was removed by filtration, and the filtrate was collected. Thesolvent was removed from the filtrate under reduced pressure to give 65g of pale yellow colored viscous liquid. The material was dried undervacuum for one week at room temperature. This was used directly for thein vitro and in vivo assay.

The following Examples were prepared using the above procedure. TABLE 2PLURONIC ® Polymers (PLU) having p-nitrophenyl phosphate lipaseinhibiting groups with a variety of hydrophobic moieties. WT. % OFHYDROPHOBIC PLU ETHYLENE MOIETY PHYSICAL EXAMPLE MW GLYCOL (R) STATE 291,100 10 wt % n-tetradecyl liquid 30 1,100 10 wt % n-dodecyl liquid 311,100 10 wt % n-decyl liquid 32 1,100 10 wt % n-octyl liquid 33 1,900 50wt % n-hexyl liquid 34 1,900 50 wt % n-octyl liquid 35 1,900 50 wt %n-decyl liquid 36 1,900 50 wt % n-dodecyl liquid 37 1,900 50 wt %n-tetradecyl semi solid 38 1,900 50 wt % n-hexadecyl semi solid 39 8,40080 wt % n-pentyl powder 40 8,400 80 wt % n-hexyl powder 41 2,900 40 wt %n-octadecyl semi solid 42 2,900 40 wt % n-hexadecyl semi solid 43 2,90040 wt % n-tetradecyl liquid 44 2,900 40 wt % n-dodecyl liquid 45 4,40040 wt % n-octadecyl semi solid 46 4,400 40 wt % n-hexadecyl semi solid47 4,400 40 wt % n-tetradecyl liquid 48 4,400 40 wt % n-dodecyl liquid

EXAMPLE 51 Preparation of a Polypropylene Glycol having a n-hexadecylHydrophobic Moiety and p-nitrophenyl Phosphate Lipase Inhibiting Group

A mixture of n-hexadecanol (28.41 g, 117 mmol) and N-methyl imidazole(9.34 mL, 117 mmol) in anhydrous methylene chloride (75 mL) was addedslowly over 20 minutes under anhydrous condition to a solution ofp-nitrophenyl phosphorodichloridate (30 g, 117 mmol) in anhydrousmethylene chloride (60 mL). The reaction flask was cooled in a waterbath during the addition. After the completion of the addition, thewater bath was removed and the reaction mixture was stirred for 2 hoursat room temperature. A mixture of polypropylene glycol (MW=1000; 58.5 g,58.5 mmol) and N-methyl imidazole (9.3 mL, 117 mmol) in anhydrousmethylene chloride (150 mL) was added to the reaction flask underanhydrous conditions. The mixture was stirred for 24 hours at roomtemperature. The reaction mixture was extracted with cold saturatedsolution of Na₂SO₄ (3×150 mL). The organic layer was dried overanhydrous magnesium sulfate. The magnesium sulfate was removed byfiltration, and the filtrate was collected. The solvent was removed fromthe filtrate under reduced pressure to give a product of 77 g. Thematerial was dried under vacuum at room temperature for 4 days.

The following polypropylene glycol p-nitrophenyl phosphates wereprepared using the above procedure. TABLE 3 Polypropylene glycol (PPG)having p-nitrophenyl phosphate lipase inhibiting groups with a varietyof hydrophobic moieties. PPG HYDROPHOBIC PHYSICAL EXAMPLE MW MOIETY (R)STATE 49 1,000 n-pentyl semi solid 50 1,000 n-octyl semi solid 51 1,000n-hexadecyl semi solid 52 1,000 n-octadecyl semi solid 53 2,000 n-pentylsemi solid 54 2,000 n-octyl semi solid 55 2,000 n-hexadecyl semi solid56 2,000 n-octadecyl semi solid

EXAMPLE 57 Preparation of a Polyethylene Glycol Polymer having ap-nitrophenyl Phosphonate Lipase Inhibiting Group and having a PentylHydrophobic Moieties A. Preparation of O,O-dimethyl n-pentylphosphonate

O,O-dimethyl phosphonate (220 g, 2 mol) was added dropwise to asuspension of NaH (48 g, 2 mol) in anhydrous THF (600 mL) undernitrogen. After 1 hour, 1-bromopentane (248 mL, 2 mol) in THF (400 mL)was added slowly, and the reaction mixture was refluxed for 12 hours.The solvent was removed under vacuum, diethyl ether (1 L) was added, andthe salts were removed by filtration. The ether solution was washed withwater (3×100 mL), the organic layer was dried over anhydrous sodiumsulfate. The ether was removed under reduced pressure, and the crudeproduct was purified by distillation under vacuum to give 171 g ofO,O-dimethyl n-pentyl phosphonate.

B. Preparation of n-pentylphosphonic dichloride

O,O-dimethyl n-pentyl phosphonate (158 g, 0.88 mol) and N,N-dimethylformamide (700 mg) were dissolved in thionyl chloride (200 mL), and theresulted mixture was refluxed for 48 hours. The volatiles were removedunder vacuum at room temperature, and the crude product was purified bydistillation to give a colorless liquid (135 g).

C. Preparation of polyethylene glycol having a p-nitrophenyl n-pentylphosphonate lipase inhibiting groups

To a solution of n-pentylphosphonic dichloride (2.65 g, 14 mmol) in 40mL of anhydrous dichloromethane, was added bright orange colored sodiumsalt of p-nitrophenol (2.3 g, 14 mmol) under anhydrous condition. Thebright orange color disappeared within 5-10 minutes. After 45 minutes, amixture of polyethylene glycol (MW=8,400; 56 g, 7 mmol) andN-methylimidazole (1.5 mL, 20 mmol) was added at room temperature andstirred for 24 hours. The reaction mixture was washed with 2% K₂CO₃solution (6×100 mL) followed by saturated NaCl solution (6×100 mL). Theorganic layer was dried over Na₂SO₄, the solvent was removed undervacuum to give a viscous liquid. The product was poured into 200 mL ofdiethyl ether and stirred for 10 minutes. The ether portion wasdecanted, and the procedure was repeated three more times. The productwas obtained as a white powder which was dried under vacuum at roomtemperature for a week.

The following polyethylene glycol polymers having p-nitrophenylphosphonate lipase inhibiting groups were prepared by this procedure.TABLE 4 Polyethylene glycols having p-nitrophenyl phosphonate lipaseinhibiting groups with a pentyl hydrophobic moiety. HYDROPHOBIC PHYSICALEXAMPLE PEG MOIETY (R) STATE 57 8,400 n-pentyl powder 58 3,400 n-pentylpowder 59 1,500 n-pentyl semi solid 60 1,000 n-pentyl semi solid

EXAMPLE 61 Preparation of a PLURONIC® Polymer having p-nitrophenylPhosphates Lipase Inhibiting Group Tethered by a n-pentyl-1,5-dioxyLinker and having a n-hexadecyl Hydrophobic Moiety

A 1 L, round-bottomed flask was charged with sodium hydride (4.0 g as a60% dispersion of NaH in mineral oil, 0.1 mol) then washed withanhydrous heptane (3×25 mL). Anhydrous tetrahydrofuran (THF) (150 mL)was added, and the suspension was stirred at room temperature undernitrogen. A solution of PLURONIC® (MW=1900, 50 wt % polyethylene glycol,50 wt % polypropylene glycol; 95 g, 0.05 mole) in anhydrous THF (200 mL)was added at room temperature. A solution of bromopentyl acetate (20.9g, 0.1 mole) in anhydrous THF (50 mL) was added to the reaction mixtureunder anhydrous conditions. The reaction mixture was refluxed at 60° C.for 16 hours. The solvent was removed under vacuum, and the resultingslurry was suspended in dichloromethane (300 mL). The solids wereremoved by filtration, and the filtrate was washed with water (3×100mL). The organic layer was dried over anhydrous sodium sulfate, and thesolvent was removed to give a pale brown viscous liquid (110 g). Thismaterial was dissolved in methanol (500 mL) and treated with aqueous 4NNaOH (40 mL). After 4 hours, the reaction mixture was acidified withconcentrated HCl, and the solvent was removed under vacuum. The viscousoil was dissolved in dichloromethane, which was washed with water (4×100mL). The organic layer was dried over sodium sulfate, and the solventwas removed to give bis-5-hydroxypentoxy PLURONIC® as a pale brownviscous liquid (98 g).

In a separate flask, a mixture of n-hexadecanol (7.02 g, 29.0 mmol) andN-methyl imidazole (2.3 mL, 290 mmol) in anhydrous methylene chloride(40 mL) was added slowly over 20 minutes under anhydrous conditions to asolution of p-nitrophenyl phosphorodichloridate (7.41 g, 29.0 mmol) inanhydrous methylene chloride (100 mL). The reaction flask was cooled ina water bath during the addition. After the completion of the addition,the water bath was removed, and the reaction mixture was stirred for 2hours at room temperature. A mixture of bis-5-hydroxypentoxy PLURONIC®(30 g, 14.48 mmol), and N-methyl imidazole (2.3 mL) in anhydrousmethylene chloride (150 mL) was added to the reaction flask underanhydrous conditions. The mixture was stirred for 24 hours at roomtemperature, then washed with saturated NaCl solution (3×100 mL). Theorganic layer was collected and dried over sodium sulfate. The solventwas removed to give a viscous liquid. This was washed with boilinghexane (6×50 mL), and the product was dried under vacuum at roomtemperature overnight to yield a pale yellow viscous liquid (39 g).

The following Examples were prepared using the above procedure. TABLE 5PLURONIC ® polymers having p-nitrophenyl phosphate lipase inhibitinggroups tethered by a variety of dialkoxys and having a variety ofhydrophobic moieties. PLU HYDROPHOBIC DIALKOXY EXAMPLE MW MOIETY (R)(Z¹) 61 1900 n-pentyl n-pent-1,5-dioxy 62 1900 n-decyl n-pent-1,5-dioxy63 1900 n-hexadecyl n-pent-1,5-dioxy 64 1900 n-pentyln-undecyl-1,10-dioxy 65 1900 n-decyl n-undecyl-1,10-dioxy 66 1900n-hexadecyl n-undecyl-1,10-dioxy

EXAMPLE 67 Preparation of a Polyethylene Glycol Polymer having ap-nitrophenyl Phosphates Lipase Inhibiting Group Tethered by an-pentyl-1,5-dioxy Linker and having a n-hexadecyl Hydrophobic Moiety

A 1 L, round-bottomed flask was charged with sodium hydride (7.67 g as a60% dispersion of NaH in mineral oil, 0.19 mol) and was washed withanhydrous heptane (3×25 mL). Anhydrous THF (200 mL) was added, and thesuspension was stirred at room temperature under nitrogen. A solution ofpolyethylene glycol (MW=1,500; 150 g, 0.1 mol) in anhydrous THF (200 mL)was added at room temperature under anhydrous conditions. The mixturewas stirred for 1 hour at room temperature, then a solution ofbromopentyl acetate (41.82 g, 0.2 mol) in anhydrous THF (100 mL) wasadded to the reaction mixture. The reaction mixture was refluxed at 60°C. for 16 hours. The solvent was removed under vacuum, and the resultingslurry was suspended in dichloromethane (300 mL). The solids wereremoved by filtration, and the filtrate was washed with water (3×100mL). The organic layer was dried over anhydrous sodium sulfate, and thesolvent was removed to give a pale brown viscous liquid (110 g). Thismaterial was dissolved in methanol (500 mL) and treated with aqueous 4NNaOH (80 mL). After 4 hours, the reaction mixture was acidified withconcentrated HCl, and the solvent was removed under vacuum. The viscousoil was dissolved in dichloromethane and was washed with water (4×100mL). The organic layer was dried over sodium sulfate, and the solventwas removed to give a bis-5-hydroxypentoxy polyethylene glycol as a palebrown viscous liquid (98 g). The p-nitrophenyl phosphate group was addedin a manner analogous to the procedure in Example 61.

The following Examples were prepared using the above procedure. TABLE 6Polyethylene glycols having a p-nitrophenyl phosphate lipase inhibitinggroup tethered by a dialkoxy linker and having a variety of hydrophobicmoieties. PEG HYDROPHOBIC DIALKOXY EXAMPLE MW MOIETIES (R) (Z¹) 67 1500n-hexyl n-pent-1,5-dioxy 68 1500 n-dodecyl n-pent-1,5-dioxy 69 1500n-hexadecyl n-pent-1,5-dioxy

EXAMPLE 75 Preparation of a BRIJ® Polymer having a p-nitrophenylPhosphate Lipase Inhibiting Group and a Hexadecyl Hydrophobic Moiety

p-Nitrophenyl phosphorodichloridate (75 g, 0.29 mol) in anhydrousdichloromethane (300 mL) was added to a 1 L, three necked,round-bottomed flask with stir bar that had been purged with N₂. Asolution of hexadecanol (71.03 g, 0.29 mol) and N-methylimidazole (23.35mL, 0.29 mol) in anhydrous dichloromethane (250 mL) was added dropwiseover a period of 2 hours. The reaction mixture was stirred for anadditional 1 hour before pouring into a 1 L separatory funnel.N-methylimidazole hydrochloride salts separated at the bottom as an oiland were removed from the funnel. Dichloromethane was removed from themixture at less than 30° C. under vacuum to give an amber oil which wastaken up in hexane (400 mL) and placed in a freezer overnight. Thereaction mixture was then thawed and the soluble portion was filtered toremove the crystals of p-nitrophenyl phosphorodichloridate. The solventwas removed from the filtrate via rotary evaporation at less than 35° C.to give n-hexyl p-nitrophenyl phosphorochloridate.

A 500 mL flask with stir bar was purged with N₂. The n-hexylp-nitrophenyl phosphorochloridate (20 g, 0.043 mol) in anhydrous THF (25mL) was added, followed by slow addition of a solution of BRIJ® 58(polyoxyethylene(20) cetyl ether; 48.56 g, 0.043 mol) andN-methylimidazole (3.45 mL, 0.043 mol) in anhydrous THF (200 mL). Thereaction mixture was stirred at room temperature for 24 hours. Thesolvent was removed at less than 35° C. by rotary evaporation, and theoily residue was dissolved in methanol (50 mL). A solution ofmethanol/water (85 mL: 15 mL, 200 mL) was added. The solidbis-n,n-dihexyl p-nitrophenyl phosphate was collected by filtration. Themethanol was then stripped off on a rotary evaporator at less than 35°C. Water was removed from the product by lyophilization.

The Examples in Table 7 can be represented by the following structureand were prepared using the above procedure. TABLE 7

BRIJ ® polymers having a terminal p-nitrophenyl phosphate lipaseinhibiting group with a variety of hydrophobic moieties. HYDROPHOBICEXAMPLE POLYMER MOIETY 70 BRIJ ® 98 (n = 19, x = 17) n-dodecyl 71 BRIJ ®98 (n = 19, x = 17) n-hexadecyl 72 BRIJ ® 35 (n = 22, x = 11) n-dodecyl73 BRIJ ® 35 (n = 22, x = 11) n-hexadecyl 74 BRIJ ® 58 (n = 19, x = 15)n-dodecyl 75 BRIJ ® 58 (n = 19, x = 15) n-hexadecyl

EXAMPLE 76 Preparation of an IGEPAL® Polymer having a Terminalp-nitrophenyl Phosphate Lipase Inhibiting Group with a n-hexadecylHydrophobic Moieties

A 500 mL flask with stir bar was purged with N₂, and n-hexadecylp-nitrophenyl phosphorochloridate (20 g, 0.043 mol) in anhydrous THF (25mL) was added, followed by slow addition of a solution of IGEPAL® 720(32.41 g, 0.043 mol) and N-methylimidazole (3.45 mL, 0.043 mol) in THF(200 mL). The reaction mixture was stirred at room temperature for 24hours. The solvent was removed under vacuum at room temperature, andoily product was taken up in methanol (50 mL). A solution ofmethanol/water (85: 15, 200 mL) was added to product. Thebis-n,n-dihexyl p-nitrophenyl phosphate was filtered off, and methanolwas then stripped off under vacuum at less than 35° C. Water was removedfrom the product by lyophilization.

The Examples in Table 8 can be represented by the following structureand were prepared using the above procedure. TABLE 8

IGEPAL ® polymers having a terminal p-nitrophenyl phosphate lipaseinhibiting group with a variety of hydrophobic moieties. HYDROPHOBICEXAMPLE POLYMER MOIETY (R) 76 IGEPAL ® 720 (n = 11) n-dodecyl 77IGEPAL ® 720 (n = 11) n-hexadecyl 78 IGEPAL ® 890 (n = 39) n-dodecyl 79IGEPAL ® 890 (n = 39) n-hexadecyl

EXAMPLE 80 Preparation of [Poly(propylene glycol) Block-poly(ethyleneglycol) Blockpoly(propylene glycol)] Polymers having a p-nitrophenylPhosphate Lipase Inhibiting Group with a n-hexyl Hydrophobic Moiety

A 500 mL flask with stir bar was purged with N₂, and n-hexylp-nitrophenyl phosphorochloridate (20 g, 0.043 mol) in anhydrous THF (25mL) was added followed by slow addition of a solution of [poly(propyleneglycol) block-poly(ethylene glycol) block-poly(propylene glycol)](average MW=2000, 50 wt. % ethylene glycol; 49.36 g, 0.0215 mol) andN-methylimidazole (3.45 mL, 0.043 mol) in THF (200 mL). The reactionmixture was stirred for 24 hours at room temperature. The solvent wasremoved under vacuum at room temperature, and the oily residue was takenup in methanol (50 mL). A mixture of 85:15 methanol:water solution (200mL) was added, and the bis-n,n-dihexyl p-nitrophenyl phosphateprecipitate was filtered off. Methanol was stripped off by rotaryevaporation at less than 35° C., and water was removed from the productby lyophilization.

The following Examples were prepared using the above procedure. TABLE 9Poly(propylene glycol) block-poly(ethylene glycol) block- poly(propyleneglycol) polymers (PPG-PEG-PPG) having p-nitrophenyl phosphate lipaseinhibiting groups with a variety of hydrophobic moieties. HYDROPHOBICEXAMPLE POLYMER MOIETY (R) 80 PPG-PEG-PPG 2000 hexyl 81 PPG-PEG-PPG 2000dodecyl 82 PPG-PEG-PPG 2000 hexadecyl

EXAMPLE 83 Preparation of a PLURONIC® Polymer having PhosphochloridateLipase Inhibiting Groups and a Decyl Hydrophobic Moiety

After purging with N₂, a solution of phophorousoxychloride (30 g, 0.1956mol) in anhydrous THF (100 mL) was added to a 3 L flask, and the mixturewas cooled to 0-5° C. A mixture of freshly distilled triethylamine(27.27 mL, 0.1956 mol) and 1-decanol (30.97 g, 0.1956 mol) in anhydrousTHF (300 mL) was added dropwise at a maximum rate of 75 mL/hour, keepingthe solution temperature at 5° C. After the addition was complete, amixture of PLURONIC® (average MW=2900, 142 g, 0.0489 mol) and freshlydistilled triethylamine (13.7 mL, 0.0978 mol) in anhydrous THF (300 mL)was added at a maximum rate of 75 mL/hour, keeping the solutiontemperature at 5° C. After the addition was complete, the reaction wasallowed to warm to room temperature and stirred for 24 hours. Thetriethylammonium hydrochloride salts were removed by filtration. Thesolvent was removed under vacuum at 30° C., and the resulting oil waswashed with hexane (6×250 mL) to remove the unreacted n-decylphosphorodichloridate. The product, bis-n-decyl phosphorochlorodatePLURONIC®, was dried under high vacuum (0.003 mm Hg) overnight at roomtemperature.

EXAMPLE 84 Preparation of a PLURONIC® Polymer havingN-hydroxysuccinimidyl Phosphate Lipase Inhibiting Groups and a DecylHydrophobic Moiety

A 125 mL flask with stir bar was purged with N₂, and a solution ofbis-n-decyl phosphorochlorodate PLURONIC® (prepared as in Example 82; 30g, 0.0178 mol) was added. N-hydroxysuccinimide (2.05 g, 0.0178 mol) wasadded as a solid and allowed to dissolve. Freshly distilledtriethylamine (2.48 mL, 0.0178 mol) was added, and the reaction mixturewas allowed to stir for 0.5 hours. The triethylammonium hydrochloridesalt was filtered off, and the THF was removed from the filtrate byrotary evaporation at 30° C. The product was dried under high vacuum(0.003 mm Hg) overnight.

EXAMPLE 85 Preparation of PLURONIC® Polymers having PyridoxinylPhosphate Lipase Inhibiting Groups and a Decyl Hydrophobic Moiety

A 125 mL flask with stir bar was purged with N₂, and bis-n-decylphosphorochlorodate PLURONIC® (prepared as in Example 82; 30 g, 0.0178mol) in anhydrous dichloromethane (30 mL) was added. Pyridoxinehydrochloride (2.54 g, 0.0178 mol) was added as a solid and allowed todissolve. Freshly distilled triethylamine (4.96 mL, 0.0356 mol) wasadded, and the reaction mixture was allowed to stir for 2 hours. Thetriethylammonium hydrochloride salt was filtered off, and the solventwas removed by a rotary evaporation at less than 35° C. The oil wastaken up in THF (50 mL) and refiltered. The solvent was removed byrotary evaporation, and the product was dried under high vacuum (0.003mm Hg) overnight at room temperature.

Table 10 lists the polymers prepared in Examples 83, 84 and 85. TABLE 10PLURONIC ® polymers having a variety of leaving groups. HYDROPHOBICLEAVING GROUP EXAMPLE POLYMER MOIETY (R) (Y-R¹) 83 PLU 2900 decylchloride 84 PLU 2900 decyl n-hydroxysuccinyl 85 PLU 2900 decylpyridoxinyl

EXAMPLE 86 Preparation of a PLURONIC® Polymer having β-Lactone LipaseInhibiting Group (Scheme I)

Intermediate 1:

10-Hydroxy methyldecanoate 1 (20 g, 98 mmol), benzyloxy2,2,2-trichloroacetimidate (30 g, 118 mmol), dichloromethane (50 mL) andcyclohexane (100 mL) were added to a 1 L, round-bottomed flask. Themixture was stirred for 5 minutes at room temperature. Trifloromethanesulfonic acid (1.3 mL) was added to the reaction mixture under nitrogenatmosphere. Within a few minutes the temperature rose from roomtemperature to 37° C. The reaction was monitored by TLC (hexane:ethylacetate; 9:1). After 16 hours, the starting material completelydisappeared. The solids were separated from the reaction by filtration,and the filtrate was washed with aqueous saturated sodium bicarbonatesolution (3×100 mL) followed by water (3×100 mL). The organic phase wascollected and dried over anhydrous sodium sulfate. The solvent wasremoved under vacuum at room temperature. The residue was purified onsilica gel column using a gradient of ether/hexane as the mobile phase.The product was eluted from the column in ether-hexane (8:2). Thesolvent was removed in vacuo to yield 10-benzyloxy methyldecanoate(intermediate 1) as a solid (32 g).

Intermediate 2:

Intermediate 1 (30 g) was saponified in 6N NaOH solution (100 mL) for 12hours, then acidified with concentrated HCl. The product was extractedwith chloroform (5×100 mL). The organic layers were combined and driedover sodium sulfate. The solvent was removed under vacuum to give10-benzyloxy decanoic acid (intermediate 2) (27 g), which was useddirectly in the next reaction.

Intermediate 3:

n-Butyl lithium in hexane (1.6 M solution, 68 mL, 108 mmol) was addeddropwise to a solution of N,N-diisoproyl amine (15.14 mL, 108 mmol) inTHF (50 mL) which was maintained at 0° C. After the completion ofaddition, the mixture was stirred for an additional 10 minutes at 0° C.The mixture was cooled to −50° C., then a solution of intermediate 2 (15g, 54 mmol) in 100 mL of THF was added dropwise. After the completion ofthe addition, the mixture was allowed to warm to room temperature, thenstirred for 1 hour. The mixture was cooled to −78° C., and a solution ofdecyl aldehyde (8.44 g, 54 mmol) in THF (40 mL) was added dropwise.After stirring for 3 hours at −78° C., the mixture was warmed to roomtemperature, then quenched by addition of saturated ammonium chloridesolution (50 mL). The mixture was extracted with diethyl ether (5×50mL). The organic layers were combined and dried over sodium sulfate,filtered and evaporated to give intermediate 3 (22 g).

Intermediate 4:

Benzenesulfonyl chloride (9.8 g, 56 mmol) was added to a solution ofintermediate 3 (12 g, 28 mmol) in pyridine (200 mL) maintained at 0° C.After addition was complete, the mixture was kept in a refrigerator at4° C. for 24 hours, then poured into crushed ice (2 kg) and stirred atroom temperature for 20 minutes. The mixture was extracted with diethylether (6×150 mL). The combined organic layers were washed with water,dried over sodium sulfate, filtered and concentrated in vacuo. Theproduct was purified on a silica gel column using hexane:ethyl acetate(9:1) to give intermediate 4 as an oil (9.8 g). IR: 1825⁻¹ cm.

Intermediate 5:

Intermediate 4 (9.5 g, 22 mmol) was dissolved in methylene chloride,then hydrogenated under 50 psi of hydrogen for 4 hours using 10% Pd/C (1g) as a catalyst. The solution was filtered, and the solvent was removedunder vacuum to give intermediate 5 as an oil (6.9 g).

Intermediate 6:

PLURONIC® (MW=1,900; 570 g; 300 mmol) in THF (500 mL) was added dropwiseto a stirred suspension of sodium hydride (15 g) in THF (150 mL). Afterthe addition was complete, the mixture was stirred for an additional 30minutes at room temperature. A solution of ethyl 4-bromobutyrate (1 17g,600 mmol) was added dropwise, and the mixture was stirred at 60° C. for16 hours. After cooling to room temperature, the salts were filteredoff, and the solvent was removed under vacuum to give light brownviscous material which was suspended in dichloromethane (1 L) and washedwith water (3×200 mL). The organic layer was collected and dried oversodium sulfate, filtered, and the solvent was removed under vacuum togive intermediate 6 as a viscous liquid (770 g).

Intermediate 7:

Intermediate 6 was dissolved in a solution of methanol (1 L) and 50%sodium hydroxide solution (100 mL), then stirred for 24 hours at roomtemperature. The reaction mixture was acidified with concentrated HCl,and the solvent was removed under vacuum. The residue was resuspended indichloromethane (1 L), then washed with water (4×250 mL). The organiclayer was dried over sodium sulfate, filtered, and the solvent wasremoved under vacuum to give intermediate 7 as a viscous liquid (650 g).

Intermediate 8:

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.8 g, 25mmol) was added under nitrogen to a solution of intermediate 7 (20.72 g,10 mmol) in dichloromethane (100 mL) in a round bottom flask. Themixture was stirred for 10 minutes at room temperature, then N-hydroxysuccinimide (2.3 g) was added. The mixture was stirred for 12 hours atroom temperature, then transferred to a separatory funnel and was washedwith water (3×30 mL). The organic layer was dried over anhydrous sodiumsulfate, filtered, and the solvent was removed under vacuum to give 22 gof intermediate 8 which was used directly in the next step.

EXAMPLE 86

Triethylamine (3 mL) was added to a solution of intermediate 8 (22 g,˜10 mmol) and intermediate 5 (6.5 g, 20 mmol) in dichloromethane (150mL). The mixture was stirred for 4 hours at room temperature, thenpoured into a separatory funnel and washed with 5% HCl (3×20 mL) andwater (3×50 mL). The organic layer was dried over sodium sulfate,filtered, and the solvent was removed under vacuum. Example 86 wasobtained as viscous liquid (26 g). This material was used directly inthe in vitro and in vivo assay.

EXAMPLE 87 Preparation of a PLURONIC® Polymer having a Disulfide LipaseInhibiting Group

EXAMPLE 87

Intermediate 9:

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (1.1 g, 5 mmol) was addedto a solution of 5,5′-dithiobis(2-nitrobenzoic acid) (3.96 g, 10 mmol)in dichloromethane (100 mL). After 10 minutes N-hydroxysuccinimide (0.5g, 5 mmol) was added, and the reaction was stirred for 6 hours at roomtemperature. The reaction mixture was poured into a separatory, thenwashed with water (3×20 mL). The organic layer was dried over anhydroussodium sulfate, filtered, and the solvent was removed under vacuum togive intermediate 9 which was used directly in the next step.

A solution of PLURONIC® (MW=1,900; 9.5 g; 5 mmol) in dichloromethane (50mL), followed by triethylamine (0.5 mL) was added to a solution ofintermediate 9 in dichloromethane (100 mL). The mixture was stirred for16 hours at room temperature, then poured into a separatory funnel andwashed with water (3×30 mL). The organic layer was dried over anhydroussodium sulfate, filtered and The solvent was removed under vacuum togive Example 87 as a viscous liquid (12 g).

EXAMPLE 88 Preparation of a PLURONIC® Polymer having an Anhydride LipaseInhibiting Group

EXAMPLE 88

Intermediate 10:

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (2.2 g, 10 mmol) was addedto a solution of 1,2,3-benzene tricarboxylic anhydride (2.1 g, 10 mmol)in dichloromethane (100 mL). The mixture was stirred for 10 minutes,then N-hydroxysuccinimide (1.0 g, 10 mmol) was added, and the reactionwas stirred for 6 hours at room temperature. The reaction mixture waspoured into a separatory funnel, then washed with water (3×20 mL). Theorganic layer was dried over anhydrous sodium sulfate, filtered, and thesolvent was removed under vacuum to give intermediate 10 which was useddirectly in the next step.

A solution of PLURONIC® (MW=1,900; 9.5 g; 5 mmol) and triethylamine (0.5mL) in dichloromethane (50 mL) was added to a solution of intermediate10 in dichloromethane (100 mL). The mixture was stirred for 16 hours atroom temperature, then poured into a separatory funnel and washed withwater (3×30 mL). The organic layer was dried over anhydrous sodiumsulfate, filtered, and the solvent was removed under vacuum to giveExample 88 (11.2 g) as viscous liquid.

In Vitro Assay

Procedure 1: Tributyrin Substrate

Potential inhibitors of pancreatic lipase activity were evaluated usinga titration method employing a pH Stat instrument (Radiometer America,Westlake Ohio). Substrate (1 mL tributyrin) was added to 29.0 mL ofTris-HCl buffer (pH 7.0) containing 100 mM NaCl, 5 mM CaCl₂, and 4 mMsodium taurodeoxycholate. This solution was stirred for 5 minutes priorto the addition of 210 units of porcine pancreatic lipase (Sigma, 21,000units/mg) dissolved in the assay buffer. The release of butyric acid bythe lipase was monitored over a 10 minute period by titrating the assaysystem to a constant pH of 7.0 with 0.02 M NaOH. Enzyme activity wasexpressed as milliequivalents of base added per minute per gram ofenzyme. In subsequent assays, varying amounts of inhibitor weresolubilized in either tributyrin or buffer, depending on the solubilitycharacteristics of the compound, and added to the assay system at timezero.

Procedure 2: Olive Oil Substrate

Potential inhibitors of pancreatic lipase activity were evaluated usinga titration method employing a pH Stat instrument (Radiometer America,Westlake, Ohio). Substrate (15 mL of an olive oil emulsion containing 80mM olive oil and 2 mM oleic acid, dissolved and sonified in a bufferconsisting of 10 mM Tris-HCl pH 8.0, 110 mM NaCl, 10 mM CaCl₂, 2 mMlecithin, 1.32 mM cholesterol, 1.92 mM sodium glycocholate, 1.28 mMsodium taurocholate, 2.88 mM sodium glycodeoxycholate, and 1.92 mMsodium taurodeoxycholate) was added to 15 mL of assay buffer (Tris-HClpH 8.0 containing 110 mM NaCl and 10 mM CaCl₂). This solution wasstirred for 4 minute prior to the addition of 1050 units of porcinepancreatic lipase (Sigma, 21,000 units/mg) dissolved in assay buffer.The hydrolysis of triglyceride was monitored over a 30 minute period bytitrating the assay system to a constant pH of 8.0 with 0.02M NaOH.Enzyme activity was expressed as milliequivalents of base added perminute per gram of enzyme. In subsequent assays, stock solutions ofinhibitor were prepared in either ethanol or DMSO, and varying amountswere added to the assay system at time zero.

The assays were conducted as described above using either procedure 1 or2, and the percent inhibition was derived by comparing the enzymeactivities in the presence and absence of inhibitor. Threeconcentrations of inhibitor were assayed, and the percent inhibition wasplotted against the log of the inhibitor concentration in order todetermine the concentration at which 50% inhibition occurred (IC₅₀). Thefollowing compounds were assayed, with the indicated values for IC₅₀presented in Tables 11-17. TABLE 11 IC₅₀ (μM) IC₅₀ (μM) with with OliveExample Polymer Hydrophobic Moiety Tributyrin Oil Polyethylene glycol(PEG) nitrophenyl phosphates: 10 PEG 1000 pentyl phosphate 400 *** 11PEG 1000 hexyl phosphate na *** 12 PEG 1000 octyl phosphate 538 *** 5PEG 1000 decyl phosphate na *** 6 PEG 1000 dodecyl phosphate 466 *** 7PEG 1000 tetradecyl phosphate 1142 *** 8 PEG 1000 hexadecyl phosphate 67320 9 PEG 1000 octadecyl phosphate 98 *** 13 PEG 1000 docosyl phosphate345 *** 14 PEG 1000 cholesteryl phosphate 172 *** 16 PEG 1500 pentylphosphate na *** 19 PEG 1500 hexadecyl phosphate 215 *** 29 PEG 1500octadecyl phosphate 73 *** 24 PEG 1500 5-phenyl-1-pentyl 24 942phosphate 22 PEG 1500 farnesyl phosphate na *** 23 PEG 1500 cholesterylphosphate 307 *** 15 PEG 3400 pentyl phosphate 559 *** 1 PEG 8400 pentylphosphate 455 *** Polypropylene glycol (PPG) nitrophenyl phosphates: 49PPG 1000 pentyl phosphate 4000 *** 53 PPG 2000 pentyl phosphate 52000*** PLURONIC ® polymers having nitrophenyl phosphate: 32 PLU 1100 octylphosphate 61 601 31 PLU 1100 decyl phosphate 174 454 30 PLU 1100 dodecylphosphate 55 400 29 PLU 1100 tetradecyl phosphate 133 1200  33 PLU 1100hexyl phosphate 155 353 39 PLU 1900 pentyl phosphate 3.6 9000  34 PLU1900 octyl phosphate 3.8 379 35 PLU 1900 decyl phosphate 2.4 105 36 PLU1900 dodecyl phosphate 2.3 183 37 PLU 1900 tetradecyl phosphate 3.6 18738 PLU 1900 hexadecyl phosphate 22 196 44 PLU 2900 dodecyl phosphate 1.7286 43 PLU 2900 tetradecyl phosphate 1.7 260 42 PLU 2900 hexadecylphosphate 0.9 106 41 PLU 2900 octadecyl phosphate 1.0 174 48 PLU 4400dodecyl phosphate 8.4 *** 47 PLU 4400 tetradecyl phosphate 5.0 *** 46PLU 4400 hexadecyl phosphate 1.4 *** 45 PLU 4400 octadecyl phosphate 4.8*** 39 PLU 8400 pentyl phosphate 325 *** 40 PLU 8400 hexyl phosphate 84*** Polyethylene glycol (PEG) nitrophenyl phosphonates: 60 PEG 1000pentyl phosphonate 836 na 59 PEG 1500 pentyl phosphonate na ***PLU = PLURONIC ®PEG = Polyethylene glycolPPG = Polypropylene glycolPLU 1,100 (10 wt % PEG monomer, 90 wt % PPG monomer)PLU 1,900 (50 wt % PEG monomer, 50 wt % PPG monomer)PLU 2,900 (40 wt % PEG monomer, 60 wt % PPG monomer)PLU 4,400 (40 wt % PEG monomer, 60 wt % PPG monomer)PLU 8,400 (80 wt % PEG monomer, 20 wt % PPG monomer)na = not active;***not tested

TABLE 12 IC₅₀ values of PLURONIC ® polymers having a p-nitrophenylphosphate lipase inhibiting group and dialkoxy linkers. HYDRO- PHOBICIC₅₀ (mM) IC₅₀ (μM) EXAM- PLU MOIETY DIALKOXY with with PLE MW (R) (Z¹)Tributyrin Olive Oil 61 1900 n-pentyl n-pent-1,5-dioxy 1.8 na 62 1900n-decyl n-pent-1,5-dioxy 1.1 289 63 1900 n-hexadecyl n-pent-1,5-dioxy1.1 278 66 1900 n-hexadecyl n-undecyl-1,10- 0.8 182 dioxy

TABLE 13 IC₅₀ values of polyethylene glycol polymers having ap-nitrophenyl phosphate lipase inhibiting group and dialkoxy linkers.HYDRO- PHOBIC IC₅₀ (μM) IC₅₀ (μM) EXAM- PEG MOIETY DIALKOXY with withOlive PLE MW (R) (Z¹) Tributyrin Oil 67 1500 n-hexyl n-pent-1,5-dioxy 71na 68 1500 n-dodecyl n-pent-1,5-dioxy 58 371 69 1500 n-hexadecyln-pent-1,5-dioxy 49 184

TABLE 14 IC₅₀ values for BRIJ ® polymers having a p-nitrophenylphosphate lipase inhibiting group. IC₅₀ HYDRO- (μM) PHOBIC IC₅₀ (μM)with EX- MOIETY with Olive AMPLE POLYMER (R) Tributyrin Oil 70 BRIJ ® 98(n = 19, x = 17) n-dodecyl *** *** 71 BRIJ ® 98 (n = 19, x = 17)n-hexadecyl  250 266 72 BRIJ ® 35 (n = 22, x = 11) n-dodecyl 1800 275 73BRIJ ® 35 (n = 22, x = 11) n-hexadecyl 1900 392 74 BRIJ ® 58 (n = 19, x= 15) n-dodecyl 1100 168 75 BRIJ ® 58 (n = 19, x = 15) n-hexadecyl 2200428

TABLE 15 IC₅₀ values for IGEPAL ® polymers having a p-nitrophenylphosphate lipase inhibiting group. HYDROPHOBIC IC₅₀ (μM) IC₅₀ (μM) EX-MOIETY with with Olive AMPLE POLYMER (R) Tributyrin Oil 76 IGEPAL ® 720n-dodecyl *** *** (n = 11) 77 IGEPAL ® 720 n-hexadecyl *** *** (n − 11)78 IGEPAL ® 890 n-dodecyl 344 148 (n = 39) 79 IGEPAL ® 890 n-hexadecyl*** *** (n = 39)

TABLE 16 IC₅₀ values for PPG-PEG-PPG polymers having p-nitrophenylphosphate lipase inhibiting groups. HYDRO- IC₅₀ IC₅₀ PHOBIC (μM) (μM)EX- MOIETY with with Olive AMPLE POLYMER (R) Tributyrin Oil 81PPG-PEG-PPG 2000 n-dodecyl 2.4 283 82 PPG-PEG-PPG 2000 n-hexadecyl 1.9384

TABLE 17 IC₅₀ values for PLURONIC ® polymers having n-hexadecylhydrophobes and a variety of leaving groups. LEAVING IC₅₀ (μM) IC₅₀ (μM)PLU GROUP with with EXAMPLE Mol.wt. (Z - R¹) tributyrin Olive Oil 832900 chloride 0.9 968 84 2900 n-hydroxysuccinyl 0.9 na 85 2900pyridoxinyl 0.09 936In Vivo Studies

Examples 8, 35, 36, 41, 42, 48, 62, 63, 67-69, 71-75, 78, 81 and 82 wereevaluated for their ability to reduce daily caloric intake by increasingthe excretion of fat in the feces, and to decrease body weight gain,relative to the control group, in normal rats over a six day period.Male Sprague-Dawley rats (five to six weeks of age) were individuallyhoused and fed ad libitum a powdered “high fat diet,” consisting ofstandard rodent chow supplemented with 15% fat (consisting of 55%coconut oil and 45% corn oil) by weight. After feeding the animals thisdiet for five days, the animals were weighed and sorted into thetreatment or control groups (6-8 animals per group, each group havingequal mean body weights). Animals were treated for six days with thetest compounds, which were added to the “high fat diet” atconcentrations (w/w) of 0.0% (control), 0.3 or 1.0 percent of the diet.

Food consumption was measured for each animal throughout the study, andwas expressed as the total amount of food consumed per animal over thesix day treatment period. On day 6, each animal was weighed, and thetotal body weight gain over the treatment period was calculated.

Rat fecal samples were collected on the final three days of the six daysof drug treatment. The samples were freeze dried and ground to a finepowder. One half gram of sample was weighed and transferred toextraction cells. Samples were extracted in an accelerated solventextractor (ASE 200 Accelerated Solvent Extractor, Dyonex Corporation,Sunnyvale, Calif.) with 95% ethanol, 5% water and 100 mM KOH. The samplewas extracted in 17 minutes at 150° C. and 1500 psi. An aliquot ofextract was transferred to a test tube containing a molar excess of HCl.The sample was then evaporated and reconstituted in a detergent solutionconsisting of 2% Triton X-1200, 1% polyoxyethylene lauryl ether and 0.9%NaCl. Fatty acids were then quantitated enzymatically with acolorimetric kit (NEFAC, Wako Chemical GmbH, Neuss, Germany).

Table 18 contains values for fecal fat/consumed fat for both control andtest animals (determined enzymatically as described above), and foodconsumption and body weight gain over 6 days as compared to controlanimals.

Calculation of Fecal Fat/Consumed Fat:

Fatty acid concentrations from the enzymatic assay are expressed asmmol/mL. The mmol/mL of fatty acid is then multiplied by the number ofmilliliters of extract generated from 500 mg of sample to give the totalmmoles of fatty acid. The value for the total mmoles of fatty acid isconverted to total milligrams of fatty acid using the average molecularweight of medium to long chain fatty acid. The value is corrected forany dilutions made during sample work-up. When results are expressed asmgs/gm of feces, the total milligrams of fatty acids is multiplied by 2.When results are expressed as total milligrams of fatty acid excreted in24 hours, the mgs/gm of feces value is multiplied by fecal weight ingrams excreted in 24 hours. When the results are expressed as excretedfat as a percentage of that consumed in 24 hours, the total weight offat excreted in 24 hours is divided by the weight of fatty acidsconsumed in over 24 hours and multiplied by 100. TABLE 18 In vivoresults of selected polymers having lipase inhibiting groups. Fecal fatTotal food Total weight Compound % of consumption change Number ClassBackbone Hydrophobe consumed % of control % of control  8 phosphate PEG1000 hexadecyl   2 ± 0.5    87 ± 2.8**   66 ± 9.8** 67 phosphate C5 PEG1500 hexyl   3 ± 0.7   97 ± 7.8  127 ± 64.4 68 phosphate C5 PEG 1500dodecyl   2 ± 0.5   99 ± 12.2   82 ± 15.4* 69 phosphate C5 PEG 1500hexadecyl   3 ± 0.8  105 ± 5.5  92 ± 8.7 35 phosphate PLU 1900 decyl 23± 5    58 ± 10**  −9 ± 17** 36 phosphate PLU 1900 dodecyl 12 ± 3   62 ±7**  16 ± 18** 42 phosphate PLU 2900 hexadecyl   13 ± 2.5**    86 ±8.6**    75 ± 16.1** 41 phosphate PLU 2900 octadecyl   15 ± 3.5**   91 ±6.2*   82 ± 6.6** 48 phosphate PLU 4400 dodecyl  4 ± 1** 96 ± 7  79 ±8** 81 phosphate PPG-PEG-PPG dodecyl 2 ± 0 92 ± 8  78 ± 11** 82phosphate PPG-PEG-PPG hexadecyl 2 ± 0 114 ± 8   129 ± 12** 62 phosphateC5 PLU 1900 decyl  12 ± 3.2   47 ± 2.6   −24 ± 13.6** 63 phosphate C5PLU 1900 hexadecyl  6 ± 1**  90 ± 5**    77 ± 13.3** 71 phosphate Brij98 hexadecyl 2 ± 1 98 ± 6 85 ± 11 72 phosphate Brij 35 dodecyl 1 ± 0 95± 5  73 ± 13** 73 phosphate Brij 35 hexadecyl 1 ± 0 106 ± 10  122 ± 17**74 phosphate Brij 58 dodecyl 2 ± 0  90 ± 5** 61 ± 14 75 phosphate Brij58 hexadecyl 1 ± 0 104 ± 8  100 ± 12  78 phosphate Igepal 890 dodecyl 1± 0 93 ± 5  72 ± 13** Control 2-3 100 100Animals were treated at a dose of 1.0%*p < 0.05**p < 0.01

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1-7. (canceled)
 8. A method for treating obesity in a mammal, comprisingthe step of orally administering to the mammal an effective amount of apolymer substituted with at least one lipase inhibiting group, whereinthe lipase inhibiting group is a boronic acid.
 9. The method of claim 8,wherein the boronic acid is a phenyl boronic acid.
 10. The method ofclaim 8, wherein the lipase inhibiting group reacts with a lipase andforms a covalent bond.
 11. The method of claim 10, wherein the lipaseinhibiting group forms a covalent bond with an amino acid residue at theactive site of the lipase.
 12. The method of claim 11, wherein thelipase inhibiting, group forms a covalent bond with an amino acidresidue that is not at the active site of the lipase.
 13. The method ofclaim 8, wherein the lipase inhibiting group is an isostere of a fattyacid.
 14. The method of claim 8, wherein the polymer is a fat-bindingpolymer.